Figure 7
Dose-effect curve of p65wt-tag and ΔNH 2 p65-tag proteins expressed separately and together by transfection in 3T3-p65ko cells. (a) 3T3-p65ko cells were transiently co-transfected with pκB-conA-LUC plasmid and both pCMV-p65wt-tag and pCMV-ΔNH2p65-tag expression vectors separately or combined in different ratios: pCMV-p65wt-tag expression vector was transfected at 1 μg/million of cells whereas pCMV-ΔNH2p65-tag expression vector was transfected at 0.5, 1 and 4 μg/million of cells (ratio p65wt/ΔNH2p65 2:1, 1:1, and 1:4, respectively). Luciferase expression was then analyzed in the whole protein extracts. Internal control of transfection was carried out by co-transfection with pSV-β-Galactosidase vector and protein concentration was also measured to normalize the data. The mean was performed with results from three different experiments and standard deviation is shown as a line on the top of the bars. (b) One hundred micrograms of nuclear protein extracts from 3T3-p65ko cells transiently transfected with pCMV-p65wt-tag and pCMV-ΔNH2p65-tag expression vectors – separately and combined in the ratio 1:4 – were subject to immunoprecipitation with the anti-FLAG tag M2 mAb. Analysis was carried out by immunoblotting with antibodies against the carboxy-terminus of p65/RelA and IκBα.